CN115612637B - 一株植物乳杆菌及其应用 - Google Patents
一株植物乳杆菌及其应用 Download PDFInfo
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- CN115612637B CN115612637B CN202210887575.4A CN202210887575A CN115612637B CN 115612637 B CN115612637 B CN 115612637B CN 202210887575 A CN202210887575 A CN 202210887575A CN 115612637 B CN115612637 B CN 115612637B
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- lactobacillus plantarum
- sxp08
- lactobacillus
- ulcerative colitis
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Abstract
本发明公开了一株植物乳杆菌及其应用，属于微生物技术领域，涉及乳杆菌。一株植物乳杆菌Lactobacillus plantarumSXp08，保藏在中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC NO.24928，保藏日期为2022年5月19日。本发明植物乳杆菌SXp08具细菌素基因达31个，抑菌范围广，可体外抑制大肠杆菌、金黄色葡萄球菌等多种细菌，具有良好的益生功能，可高密度培养，能有效改善溃疡性结肠炎小鼠的肠道菌群结构，缓解DSS诱导的溃疡性结肠炎。
Description
技术领域
本发明属于微生物技术领域，涉及乳杆菌，具体涉及一株植物乳杆菌及其在调节肠道菌群中的应用。
背景技术
人体肠道内所含的微生物数量超过10万亿个，约占人体微生物总量的78.68%，数量巨大的肠道微生物共同组成了人体肠道微生态系统。该系统与人体健康息息相关，可从消化、营养吸收、能量供应、脂肪代谢、抗病等多方面影响宿主的生理功能，不仅可以帮助宿主消化食物、代谢药物以及外源化合物、合成维生素、抑制病原体的侵袭，还可影响免疫系统发育和大脑活动，甚至可以调控宿主的情绪行为。
越来越多的研究结果表明，肠道微生态系统的平衡对宿主健康起重要作用。当肠道微生物菌群结构失衡时，部分肠道微生物会对宿主的健康产生负面影响，而且众多人体疾病的病发与肠道微生物的异常相关联。如心脑血管疾病、肥胖、糖尿病、炎症性肠炎（IBD）、肠易激综合症（IBS）、胃肠道癌症、肝硬化、慢性肾病等病症的发生都与肠道微生物的异常有关。
为了保持肠道微生态系统的平衡，抑制肠道有害菌的生长，本发明筛选了一株植物乳杆菌（Lactobacillus plantarum），以达到上述目的。
发明内容
本发明的目的是提供一株植物乳杆菌。
本发明的再一个目的是提供植物乳杆菌的的应用。
本发明是通过以下技术方案实现的：
一株植物乳杆菌Lactobacillus plantarumSXp08，保藏在中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC NO. 24928，保藏日期为2022年5月19日。
所述的植物乳杆菌SXp08在制备抑菌、防腐制品中的应用。
所述的植物乳杆菌SXp08在制备调节肠道菌群的药品、和/或保健品、和/或饲料中的应用。
所述的植物乳杆菌SXp08在制备治疗溃疡性结肠炎药物中的应用。
进一步地，在上述应用中，包括了植物乳杆菌SXp08活菌体的的培养物、菌悬液或裂解物。
更进一步，在上述应用中，所述的活菌体数≥1×1010CFU/mL。
本发明通过碳酸钙溶钙圈筛选得到植物乳杆菌SXp08，具有很好的耐酸、耐胆盐胁迫能力和较强的抗氧化、分解胆固醇的益生功效，其16s rRNA基因序列在GeneBank中进行比对，结果显示为植物乳杆菌，命名为植物乳杆菌（Lactobacillus plantarum）SXp08，将SXp08接种在MRS液体培养基中，在30~37℃下培养18~24 h，提取活菌体进行细菌基因组测序分析，发现其具有30种、31个细菌素基因，无CARD抗生素抗性基因和VFDB细菌毒力因子基因，其可用于制备调节肠道菌群的药品、和/或保健品、和/或饲料。
通过抑菌圈实验，发现植物乳杆菌培养液、细胞裂解液、培养上清液对大肠杆菌、金黄色葡萄球菌、枯草芽孢杆菌、沙门氏菌、克雷伯氏菌等不同菌株具有明显的抑制效果；其可用于制备抑菌、防腐制品。
使用ICR小鼠，通过饮用葡聚糖硫酸钠建立溃疡性结肠炎模型, 比较正常对照组、模型对照组、植物乳杆菌SXp08治疗组和美沙拉嗪治疗组小鼠的肠道菌群结构，以小鼠粪便为样本，提取DNA后进行16S rDNA V3~V4区高通量测序。统计不同类型小鼠肠道菌群在门水平和属水平上的分布，植物乳杆菌具有增加厚壁菌门、毛螺菌科 NK4A136 group的相对丰度，降低变形菌门、疣微菌门、拟杆菌属和Akkermansia的相对丰度的作用；正常对照小鼠的肠道微生物平均OTU为685.6，结肠炎模型小鼠为630.5，植物乳杆菌SXp08治疗组为641，美沙拉嗪治疗组为462.8，OUT数越多代表微生物种类越多，植物乳杆菌具有维持肠道微生物多样性的作用，其可以用于制备调节肠道菌群的药品、和/或保健品、和/或饲料。
使用ICR小鼠，通过饮用葡聚糖硫酸钠建立溃疡性结肠炎模型, 比较正常对照组、模型对照组、植物乳杆菌治疗组和美沙拉嗪治疗组小鼠的体重变化、结肠长度、临床症状等方面的差异，发现植物乳杆菌具有改善溃疡性结肠炎的作用，其可以用于制备治疗溃疡性结肠炎的药物。
综上所述，本发明植物乳杆菌SXp08活菌体、发酵液、菌悬液、上清液作为主要成分，可应用于制备各种抑菌产品、调节肠道菌群和改善溃疡性结肠炎的药物、保健品、复合益生菌粉、饲料等，其中植物乳杆菌SXp08的有效活菌数≥1×1010CFU/mL。使用时，可以配以辅料，根据作用对象和用途不同，辅料可以为但不限于各种益生元、保护剂、填充剂、粘合剂、润湿剂、崩解剂、润滑剂、矫味剂、香味剂、着色剂、包衣剂、酸度调节剂、防腐剂、稀释剂、悬浮液稳定剂等。
与现有技术相比，本发明植物乳杆菌SXp08具细菌素基因达31个，抑菌范围广，可体外抑制大肠杆菌、金黄色葡萄球菌等多种细菌，具有良好的益生功能，可高密度培养，能有效改善溃疡性结肠炎小鼠的肠道菌群结构，缓解DSS诱导的溃疡性结肠炎。
附图说明
图1 植物乳杆菌SXp08不同培养温度时的生长曲线。
具体实施方式
为进一步阐述本发明所采取的技术手段及其效果，以下结合实施例对本发明作进一步地说明。此处所描述的具体实施方式仅仅用于解释本发明，而非对本发明的限定。
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购获得的常规产品。
实施例1
从山西维尔生物乳制品有限公司生牛初乳储藏罐中收集残余牛乳于无菌三角瓶中，用灭菌生理盐水将牛乳样品稀释至10-5，取原液、10-1、10-3、10-5各0.1 mL涂布MRS碳酸钙培养基平板，35 ℃厌氧培养盒培养48h，挑选溶钙圈比较大的单菌落，重复划线分离，挑选单菌落用200 mL/L甘油管-80 ˚C 保藏备用；进一步进行耐酸、耐胆盐、抗氧化能力、分解胆固醇能力的筛选以及菌株的鉴定。
筛选得到的植物乳杆菌Lactobacillus plantarum SXp08，于2022年5月19日保藏在中国微生物菌种保藏管理委员会普通微生物中心（CGMCC），地址为北京市朝阳区北辰西路1号院3号，邮编100101，保藏编号为CGMCC NO.24928。
MRS液体培养基：蛋白胨 10 g /L、牛肉浸膏 5 g /L、酵母浸膏 4 g /L、葡萄糖20 g /L、磷酸氢二钾 2 g /L、乙酸钠 5 g /L、柠檬酸三铵 2 g /L、硫酸镁 0.2 g /L、硫酸锰 0.05 g /L、吐温 80 1.0 mL/L，pH6.5，用蒸馏水定容至 1000 mL，121 ℃灭菌 15min。
MRS固体培养基：MRS液体培养基中加入20 g/L 的琼脂粉，121 ℃灭菌 15 min。
将植物乳杆菌Lactobacillus plantarum SXp08进行耐酸、耐胆盐、抗氧化能力、分解胆固醇能力的测定，测定参照已有方法进行。
菌株的生理生化鉴定参照《常见细菌鉴定手册》和《微生物分类学》，观察各菌株的形态特征，并对菌株进行生理生化实验，对筛选菌株进行初步的分类鉴定。分子生物学鉴定由金唯智公司进行16S rDNA序列测定，将测得的DNA序列提交到GenBank美国国家生物技术信息中心(National Center of Biotechnology Information, NCBI)进行比对。
植物乳杆菌SXp08的菌落直径约1～3 mm左右，凸起，呈圆形，表面光滑，细密，白色；菌体形态呈短杆状、成短链，革兰氏染色为阳性，无芽孢，产酸，兼性厌氧。
实验结果为：植物乳杆菌SXp08通过碳酸钙溶钙圈筛选，pH 2.5条件下处理2h耐酸生长率为63.72%，耐3g/mL胆盐生长率为53.28%；在菌体浓度达到1010 CFU/mL的情况下，DPPH·清除率为43.5%，羟自由基清除率为86.7%，胆固醇分解能力为51.4%。
植物乳杆菌SXp08过氧化氢酶、氧化酶、淀粉水解、明胶液化试验结果呈阴性，甲基红实验结果呈阳性。
植物乳杆菌SXp08 16s rRNA基因核酸序列：
TTTGAACTGAACAAAGTTTCGACGAATCAAATGTGTAGGGTCTCTTGATTTTGAATCAAGAGCAAAACATTTGCGAAGTCAATTCGCTAGCAAATAAATTTTAGTACAACATTAAATGAGCTTTTTAGAACTCATCATTAATTTGAGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGAACTCTGGCATTGATTGGTGCTTGCATCATGATTTACATTTGAGTGAGTGGCGAACTGGTGAGTAACACGTGGGAAACCTGCCCAGAAGCGGGGGATAACACCTGGAAACAGATGCTAATACCGCATAACAACTTGGACCGCATGGTCCGAGTTTGAAAGATGGCTTCGGCTATCACTTTTGGATGGTCCCGCGGCGTATTAGCTAGATGGCGAGGTAACGGCTCACCATGGCAATGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAACTCTGTTGTTAAAGAAGAACATATCTGAGAGTAACTGTTCAGGTATTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGTATGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCATACCGTAAACGATGAATGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATACTATGCAAATCTAAGAGATTAGACGTTCCCTTCGGGGACATGGATACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATCAGTTGCCAGCATTAAGTTGGGCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAACTCGCGAGAGTAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGTCGGTGGGGTAACCTTTTAGGAACCAGCCGCCTAAGGTGGGACAGATGATTAGGGTGAAGTCGTAACAAGGTAGCCGTAGGAGAACCTGCGGCTGGATCACCTCCTTTCTAAGGAATATTACGGAAACCTACACACGCGTCGAAACTTTGTTTAGTTTTGAGAGATTTAACTCTCAAAACTTGTTCTTTGAAAACTAGATAATATCAAATATATTTTTTCATAATGAAACCGAGAACACCGCGTTTTTTGAGTTTTATGTAGGAACCAGCCGCCTAAGGTATCGA。
实施例2
对实施例1分离得到的植物乳杆菌SXp08的生长能力进行测定，步骤为：将活化好的植物乳杆菌接种于MRS液体培养基中,设定温度下静置培养，每间隔 2 h 取样测定培养液pH和 OD600 值，培养温度范围为30~37℃，培养时间范围为18~24 h，绘制菌体生长曲线图，从图1可以看出在培养温度35℃、培养时间18h时，植物乳杆菌SXp08的菌体密度OD600 值可高达1.96，培养皿活菌计数为5.6×1011 CFU/mL，具有高密度培养的潜质。
实施例3
通过植物乳杆菌SXp08的基因组测序及抑菌圈实验考察植物乳杆菌SXp08的抑菌范围和抑菌效果。
抑菌圈实验通过在培养基上打孔进行，以抑菌圈的大小评价其抑制各种菌生长的能力，其中分别测定了包括植物乳杆菌培养液、细胞裂解液、培养上清液对不同菌株的抑制效果。
其中细菌素基因见表1，同时通过与CARD抗性基因数据库和VFDB细菌毒力因子数据库比对，植物乳杆菌SXp08无CARD抗生素抗性基因和VFDB细菌毒力因子基因，为安全的益生菌菌株。其抑菌作用见表2，从表2可以看出植物乳杆菌SXp08对大肠杆菌、金黄色葡萄球菌、枯草芽孢杆菌、沙门氏菌、克雷伯氏菌等不同菌株具有明显的抑制效果，抑菌范围广，效果明显。
表1 植物乳杆菌SXp08的细菌素基因
表2 植物乳杆菌SXp08对不同菌株的抑制效果
| 被抑制菌种 | 抑菌效果（抑菌圈直径/mm） |
| 大肠杆菌 | 16.22 |
| 金黄色葡萄球菌 | 15.38 |
| 枯草芽孢杆菌 | 17.83 |
| 沙门氏菌 | 16.91 |
| 克雷伯氏菌 | 14.56 |
| 化脓性链球菌 | 16.88 |
| 分支杆菌 | 12.75 |
| 嗜血杆菌 | 13.74 |
实施例4
将24只雄性ICR小鼠根据随机数字表法分为4组:正常对照组（CK）、模型对照组（D）、植物乳杆菌SXp08治疗组（D+P）和美沙拉嗪治疗组（D+A）,每组各6只。模型对照组和两个治疗组通过饮用葡聚糖硫酸钠建立溃疡性结肠炎模型，两个治疗组小鼠分别给予植物乳杆菌SXp08和美沙拉嗪灌胃治疗，正常对照组和模型对照组给予0.9%氯化钠溶液灌胃。14天后取各小鼠的粪便样本、提取DNA，通过16S rDNA V3~V4区高通量测序，考察植物乳杆菌SXp08对溃疡性结肠炎小鼠肠道菌群的调节作用。
4组小鼠的溃疡性结肠炎小鼠肠道微生物多样性的结果见表3，其中OUT数越多代表微生物种类越多，从表3可以看出，植物乳杆菌SXp08治疗组的肠道微生物多样性更丰富。
表3 OTU代表的小鼠肠道微生物多样性
| 小鼠模型 | CK | D | D+P | D+A |
| 肠道微生物平均OTU | 685.6 | 630.5 | 641 | 462.8 |
4组溃疡性结肠炎小鼠肠道微生物菌群结构的结果见表4和表5， 从表4和5可以看出，在门水平上，植物乳杆菌SXp08治疗组（D+P）与模型对照组（D）比较，可以增加厚壁菌门的丰度，降低拟杆菌门、变形菌门、柔膜菌门的丰度；与美沙拉嗪治疗组（D+A）比较可以显著增加厚壁菌门的丰度，降低拟杆菌门、变形菌门、放线菌门、疣微菌门的丰度。在属水平上，植物乳杆菌SXp08治疗组（D+P）与模型对照组（D）比较，可显著提高毛螺菌科NK4A136group、Lachnoclostridium、Ruminiclostridium 9、阿克曼菌属的丰度，降低拟杆菌属的丰度；与美沙拉嗪治疗组（D+A）比较可显著提高毛螺菌科NK4A136 group、Lachnoclostridium、Ruminiclostridium 9的丰度，降低拟杆菌属和阿克曼菌属的丰度。植物乳杆菌SXp08的这些对肠道菌群的调节作用对维护正常的肠道微生态系统是非常重要的。
表4 不同处理小鼠肠道菌群在门水平上的分布
表5 不同处理小鼠肠道菌群在属水平上的分布
实施例5
将实施例4的4组小鼠分别进行体重变化、结肠长度、临床观察的的相关表现进行试验，结果见表6，从表6可以看出，植物乳杆菌SXp08治疗组（D+P）的体重变化不大，而模型对照组（D）和美沙拉嗪治疗组（D+A）的体重都有所降低；植物乳杆菌SXp08治疗组（D+P）的结肠长度大于模型对照组（D）和美沙拉嗪治疗组（D+A）；临床反应优于模型对照组（D）和美沙拉嗪治疗组（D+A）。说明植物乳杆菌SXp08对缓解小鼠溃疡性结肠炎具有一定的作用。
表6 不同处理小鼠溃疡性结肠炎相关表现
实施例6
一种含有植物乳杆菌SXp08酸奶发酵剂的制备，制备方法如下：将植物乳杆菌接种于MRS液体培养基中，35℃下培养24 h进行活化，连续活化2次，得到活化液；将活化液按2%（v/v）的接种量接种于MRS液体培养基中，37℃下培养24 h，得到菌液；将菌液在8000 g下离心10 min，得到植物乳杆菌菌体；将菌体用质量浓度为10%的脱脂奶粉重悬至浓度为1×1010CFU/mL，得到菌悬液；将菌悬液在37℃下预培养1 h后冻干，得到植物乳杆菌SXp08发酵剂。在制备酸奶的时候按1.5g/100mL接种于鲜牛乳中，同时按2g/100mL接种保加利亚乳杆菌和嗜热链球菌发酵剂，由于植物乳杆菌SXp08优良的抑菌作用，使用此种方法制备的活菌酸奶冷藏保质期可延长5天。
实施例7
一种含有植物乳杆菌SXp08复合益生菌粉的制备，制备方法如下：植物乳杆菌SXp08经斜面活化后，接种于10 L种子罐，35 ℃培养16 h，按接种量20 mL/L接种于100 L发酵罐中，起始pH 6.5、培养温度35 ℃、溶氧0 mL/L（控制在100 mL/L以下）、搅拌转速50 r/min、罐压0.03 Mpa、发酵培养20 h；发酵结束后，将发酵液通过运输管道打入管式离心机中，10000 r/min连续离心20 min制备菌泥；以低聚果糖5g/100 mL、脱脂乳粉12.5g/100mL、半乳糖4.5g/100 mL、乳糖 3g/100 mL、脯氨酸2.5g/100 mL、水溶性海藻糖3g/100 mL为冻干保护剂制备植物乳杆菌冻干份；该冻干粉活菌数可达1011 CFU/g。植物乳杆菌SXp08冻干粉可复配乳双歧杆菌、植物乳杆菌、嗜酸乳杆菌、鼠李糖乳杆菌、副干酪乳杆菌等益生菌，乳糖醇、低聚木糖、赤藓糖醇、甜橙粉、异构化乳糖液等益生元和辅助材料制备复合益生菌粉。
Claims (7)
1. 一株植物乳杆菌（Lactobacillus plantarum）SXp08，保藏在中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC NO. 24928，保藏日期为2022年5月19日。
2.权利要求1所述的植物乳杆菌SXp08在制备抑菌制品中的应用，其中所述的抑菌制品为抑制大肠杆菌、金黄色葡萄球菌、枯草芽孢杆菌、沙门氏菌、克雷伯氏菌、化脓性链球菌、分支杆菌、或嗜血杆菌的制品。
3.根据权利要求2所述的应用，其特征在于，所述的植物乳杆菌SXp08为活菌体，所述的抑菌制品中植物乳杆菌SXp08的活菌数≥1×1010CFU/mL。
4.权利要求1所述的植物乳杆菌SXp08在制备调节肠道菌群的保健品、或饲料中的应用。
5.根据权利要求4所述的应用，其特征在于，所述的植物乳杆菌SXp08为活菌体，所述的保健品、或饲料中植物乳杆菌SXp08的活菌数≥1×1010CFU/mL。
6.权利要求1所述的植物乳杆菌SXp08在制备治疗溃疡性结肠炎药物中的应用。
7.根据权利要求6所述的应用，其特征在于，权利要求1所述的植物乳杆菌SXp08为活菌体，所述的药物中植物乳杆菌SXp08的活菌数≥1×1010CFU/mL。
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